The present disclosure relates generally to the assessment of the health of living tissue and the profiling of the phenotypic response of tissue to perturbations, both environmental and pharmaceutical.
Cellular systems are highly complex, with high redundancy and dense cross-talk among signaling pathways. [1] (Note: Bracketed numbers refer to general reference publications listed at the end of the disclosure). Biochemical target-based high-content screening (HCS) can isolate single mechanisms in important pathways, but often fails to capture integrated system-wide responses. Phenotypic profiling, on the other hand, presents a systems-biology approach that has more biological relevance by capturing multimodal influence of therapeutics. [2] Although phenotypic profiling predates genomics that provided isolated targets, it remains today one of the most successful approaches for the discovery of new drugs. [3]
At present most phenotypic profiling is performed on two-dimensional culture, even though two-dimensional monolayer culture on flat hard surfaces does not respond to applied drugs in the same way as cells in their natural three-dimensional environment. This is in part because genomic profiles are not preserved in primary monolayer cultures. [4-6] There have been several comparative transcriptomic studies that have tracked the expression of genes associated with cell survival, proliferation, differentiation and resistance to therapy that are expressed differently in 2D cultures relative to three-dimensional culture. For example, three-dimensional culture from cell lines of epithelial ovarian cancer [7, 8], hepatocellular carcinoma [9-11] or colon cancer [12] display expression profiles more like those from tumor tissues than when grown in 2D. In addition, the three-dimensional environment of 3D culture presents different pharmacokinetics than 2D monolayer culture and produce differences in cancer drug sensitivities. [13-16]
An alternative approach to imaging form is to image function, and in particular functional motions. Motion is ubiquitous in all living things and occurs across broad spatial and temporal scales. At one extreme, motions of molecules during Brownian diffusion occur across nanometers at microsecond scales, while at the other extreme motions of metastatic crawling cells occur across millimeters taking many hours. As one spans these scales, many different functional processes are taking place: molecular diffusion, molecular polymerization or depolymerization of the cytoskeleton, segregation of enzymes into vesicles, exocytosis and endocytosis, shepherding of vesicles by molecular motors, active transport of mitochondria, cytoskeletal forces pushing and pulling on the nucleus, undulations of the cell membrane, cell-to-cell adhesions, deformation of the cell, cell division and ultimately to movement of individual cells through tissue. All of these very different types of cellular dynamics can be active and useful indicators of the functioning behavior of cells. The functional response of target cells to applied drugs is of particular relevance in drug screening.
Imaging motion in three-dimensional tissue is simpler than imaging structure or performing molecular imaging, because motion modulates coherent light through phase modulation. When light scatters from an object that is displacing, the phase of the light is modified. If the light has coherence, then the motion-induced phase shifts of one light path interfere with the phase shifts of other light paths in constructive and destructive interference. Even light that is multiply scattered in tissue carries a record of the different types of motions that the light encountered. By measuring the fluctuating phase of light scattered from living tissue, the different types of motion across the different space and time scales can be measured. The trade-off for greater depth of penetration into three-dimensional tissue is reduced spatial imaging resolution. But volumetric imaging of intracellular motions in tissue is still possible, within limits, by using low-coherence interferometry that can select light from specified depths by using coherence-gating approaches [17].
The 2D imaging techniques have been applied to drug screening and in particular to evaluating efficacy of a drug in disease treatment, such as anticancer drugs. Traditional 2D techniques can lead to false positives when a drug is more effective in 2D than in 3D, resulting in promising early drug leads that fail in animal models because the drug is more effective at killing tumor cells grown as monolayer cultures than as cells within multicellular tissues. An even greater impact is the false negative in drug screening in which a drug that would otherwise be effective in 3D yields a negative result at the 2D screening stage, thereby leading to the elimination of the drug. The end result of false positives and false negatives is that new drug discovery halves every nine years (in contrast to Moore's Law of the electronics industry in which chip capacity doubles every 18 months).
Phenotypic profiling can provide a significant advance in drug screening when based on 3D cultures. What is needed is a system and method for extracting high-content phenotypic responses from inside heterogeneous tissue and for accurately and meaningfully analyzing the resulting information to build a phenotype database to improve the efficiency of future drug screening.